This proposal is a combined R21/R33 application to develop an integrated multidimensional analysis system for characterization of complex mixtures of proteins, i.e. proteome analysis. The system will employ a microfluidic implementation of a 2D chromatography separation system directly interfaced via electrospray ionization to a mass spectrometer. The analytical approach is to digest the protein mixture prior to separation and then separate and analyze the resulting fragment peptides. Protein identification is done by computer matching of tandem mass spectra with predicted data from sequence databases. The separation system, which will employ strong cation exchange and reversed phase partition chromatography, is based upon a published capillary system which has been shown to be superior to conventional two dimensional gel electrophoresis methods for proteomic studies in yeast. The microchip implementation will enable improvements over the published approach to yield a more powerful, yet simpler to use and less expensive system. The ultimate implementation of the system will replace 2D gel separation with a relatively inexpensive, disposable injection molded plastic microfluidic device which interfaces directly to the mass spectrometer. The prototype devices will be produced using recently developed soft lithography methods which obviate the need for complex microfabrication facilities and yield designs directly transferable to large scale production by injection molding. This approach will facilitate the application of proteome analysis in cancer research by significantly reducing the labor and art required compared to 2D electrophoresis and eliminating the need for manual transfer and processing of gel for mass spectrometric analysis. The system will facilitate the molecular analysis of cancer by providing a readily accessible approach to determining the expression patterns of proteins in normal and neoplastic tissues as well as characterizing the protein content of clinical specimens. The fact that the system will be implemented as a microfluidic device makes it amenable to very small sample sizes and is potentially extendable to single cell analysis.